Clinical symptoms, temperature, and oxygen saturation were extracted from medical records, 7 days before to 14 days after testing. COVID-19 was confirmed in 81 (21%) residents; 36 (44%) S-based and 45 (56%) TP-based: 45. Cycle threshold (CT) values did not differ between the groups. In the 7 days prior to the test falling (32%), somnolence (25%) and fatigue (21%) occurred in both groups. Two days before the test, we observed a stronger decrease in oxygen saturation and an increase in temperature for the S-based group compared to the T-based group that remained up to 10 days after testing. Residents within the S-based group were 2.5 times more likely to increased mortality within 30 days than residents in the TP-based group . Although, 73% of the T-based group did eventually develop core symptoms. Thus, attention to falling and daily measures of temperature and oxygen saturation can contribute to earlier detection.Nursing homes (NH) residents with COVID-19 can either be tested because of presence of core symptoms (S-based) or because of transmission prevention (TP-based). The investigated study sample included all NH residents who underwent SARS-CoV-2 RT-PCR testing between March 16, 2020 and May 31, 2020 ( Residents in nursing homes (NH) account for a disproportionate number of COVID-19 related deaths, making up half of all COVID-19–related deaths in Western countries . This diFurthermore, self-reporting of complaints is often compromised in residents due to limited ability to communicate  . Not be. TherefoThis study, in which NH residents were tested either because of presence of core symptoms (symptom-based testing) or because of transmission prevention (transmission–prevention-based testing), aims to describe the clinical presentation and course of COVID-19  in both groups and differences between both groups. A second aim is to compare the cycle threshold (CT) value of the RT-PCR test between the two groups.This study was conducted in residents living in the NH Amsta , a 1185-bed skilled nursing facility with 18 locations spread over [Amsterdam].In this retrospective cohort study, all residents who underwent a RT-PCR test from March 16, 2020 (the day we started testing), up to May 31, 2020 were included. RT-PCR tests were performed with a throat and nasopharyngeal swab. Patients who had a positive RT-PCR test were considered to be COVID-19 confirmed . The residents where the test was performed were divided into two groups: 1) The symptom-based test group (S-based), a test was performed when a resident had core symptoms according to the Dutch guidelines (fever (temperature ≥38.0°C/≥100.4°F), cough, or dyspnea) . 2) The Data about age, gender, BMI, type of ward, comorbidities, and renal function of all residents that were tested on COVID-19 were extracted from the electronic health record (EHR) PinkRoccade Healthcare myCaress (myCaress). Data were pseudonymized and considered as part of the usual care data; therefore, no additional informed consent were requested.For all residents with a confirmed COVID-19, we searched for clinical symptoms and for temperature and oxygen saturation data in the EHR from 7 days before testing up to 14 days after testing. The oxygen saturation was measured with a pulse oximeter and the body temperature was measured differently per ward, rectal or tympanic. The same measuring instrument was used for a resident in consecutive measurements. Both saturation and temperature were measured daily at a fixed time, mostly in the morning, as much as possible. If several measurements were registered, the first recorded measurement was used.Clinical symptoms were extracted by reviewing the EHR, using registrations of physicians as well as of nursing staff. We searched for clinical symptoms that were new or significantly changed  for each individual resident. A symptom was regarded as new if it had not been reported in the previous 4 weeks, and changed if it significantly changed compared to the previous 4 weeks.From the day that the first patient tested positive, it was agreed for the whole NH Amsta to measure the temperature and the oxygen saturation daily in all residents and to register it in the Electric Medical Record (EMR) (specific sections). Data about temperature and oxygen saturation were extracted from these specific sections. Data on 30-day mortality were collected from the EHR of individual residents, starting from the first day of the positive test.p-value of <.05 to be statistically significant. Survival curves on 30-day mortality were estimated based on the days between the positive RT-PCR test and the date of death using Kaplan–Meier curves in residents with confirmed COVID-19 (symptom-based testing and transmission prevention-based testing). The mortality rate of residents with confirmed COVID-19 (symptom-based testing vs. transmission prevention-based testing) was compared using a Cox proportional hazard model, using three models. Model 1 unadjusted, model 2 included gender and age, model 3 included gender, age, and reduced renal function. Reduced renal function was significant in the univariate analysis and was much more common in the S-based group. Results are presented with 95% confidence intervals and all reported p values are two-sided.Descriptive analysis was performed on patient demographic and symptom variables (temperature and oxygen saturation). To indicate the number of missing values, the N on which percentages were calculated are reported. All continuous data were presented as means with standard deviation and 95% confidence intervals (CI). To evaluate the differences in clinical symptoms, temperature, and oxygen saturation between the two groups of residents with confirmed COVID-19 (symptom-based testing vs. transmission prevention-based testing) group ANOVA’s were applied. We considered a Finally, using ANOVA, we compared the CT value of both groups (symptom-based testing vs. transmission prevention-based testing).All analyses were performed with the use of the SPSS statistical package, version 26.0 .On March 16, 2020, the date the first RT-PCR SARS-CoV-2 test took place, Amsta had a bed capacity for 1185 residents. Between March 16th and May 31st, 2020, 380 residents underwent a RT-PCR test for COVID-19 (601 tests). COVID-19 was ruled out in 299 (79%) residents and confirmed in 81 (21%) of residents. Of these 81 residents, 36 (44%) residents were tested because of presence of core symptoms (S-based), compared to 45 (56%) residents who tested positively but were tested because of transmission prevention (TP-based) see Figure 1.SD ± 11.2) in the S-based group and 82 years (SD ± 8.7) in the TP-based group. There were more women in both groups, S-based (53%) and TP-based (60%). In residents who were tested but for whom COVID-19 was ruled out the mean age was 79 years (SD ± 11.1), and the majority were woman 62%. Almost all residents in the TP-based group had dementia (96%), whereas in the S-based group this was 83%. In residents tested with COVID-19 ruled out, just over half of these residents had dementia (59%). After dementia, cardiac problems, hypertension, and a reduced kidney function were the most common comorbidities in residents who tested positive or negative for COVID. (Mean age for the COVID-19 residents was 80 years (r COVID. .Table 1.n = 27) had a median CT value of 24.8 (range 15.6–39), transmission prevention-based testing residents (n = 37) had a median CT value of 24.9 (range 12.5–40.4). There were no significant differences between these groups F (1.63) = .011, p = .919.Cycle Threshold values were available for 64/81 positive residents; the mean CT value was 26.2 (range 12.5–40.4). Symptom-based tested residents , rhinorrhea (33%), dysphagia (25%), body weakness (19%), and diarrhea (19%).SD ± 2.3).Fever is the most common symptom in the 14 days after the test, the majority of residents with a fever were in the S-based group (76% vs. 53% TP-based). The core symptoms cough and dyspnea were also most common in the S-based group compared to the TP-based group . Of all residents with confirmed COVID-19 tested based on transmission prevention, 73% eventually developed core symptoms after an average of 4.2 days . Agitation and confusion were more often reported in the TP-based group compared to the S-based group (agitated 31% vs. 6% and confusion 36% vs. 11%). Body weakness and anxiety were reported more often in the S-based group (body weakness 47% vs. 24% and anxiety 28% vs. 9%). Furthermore, the following symptoms were reported: dysphagia and somnolence, both more often in the S-based group, 25% and 31%, respectively. Diarrhea, fatigue, and nausea were more frequently present in the TP-based group, 24%, 27%, and 18%, respectively.The mean temperature of the residents with confirmed COVID-19 was lower in the 7 days prior to the positive RT-PCR test compared to the day of the RT-PCR test or up to ∼10 days after positive RT-PCR testing . Up to tThe mean oxygen saturation by the COVID-19 confirmed group was higher in the 7 days prior to the positive RT-PCR test compared to the day of the RT-PCR test or up to ∼10 days after positive RT-PCR testing. The oxygen saturation dropped 2 days before the positive RT-PCR test. The oxygen saturation fluctuated until approximately 11 days after the positive confirmed COVID-19 test, after which it slowly increased again . Overallp = .010). This difference remained when adjusted for gender and age  and when adjusted for gender, age, and reduced renal function .Of the residents with a confirmed COVID-19, 40% died within 30 days  More residents in the S-based group died 56%  compared to residents in the TP-based group 27%  . ResidenWe are the first to show day-to-day fluctuations of the oxygen saturation in COVID-19 positive NH residents; oxygen saturation decreases approximately two days before testing in all residents who tested positive. We observed that oxygen saturation was lower for the S-based group prior to test than for the TP-based group. In addition, we observed an increase in temperature ∼3 days prior to test. Day-to-day fluctuations in temperature in residents with COVID-19 were described previously . Yet, ouFurthermore, measuring the temperature and oxygen saturation on a daily basis is non-invasive and does not depend on the ability to express symptoms. Many NH residents frequently have difficulty putting their symptoms into words and therefore carry a high risk of not being recognized as COVID-19 patients. For example, our results demonstrated that almost all residents who were tested based on transmission prevention were living in a psychogeriatric ward and had dementia. By measuring daily changes in their temperature and oxygen saturation, and acting on this through testing and early quarantine, we may reduce the chance of transmission in this vulnerable group of NH residents. Of the residents with confirmed COVID-19, 44% of the residents were tested based on the presence of core symptoms (S-based) and 56% of the residents were tested based on transmission prevention (TP-based).In the 7 days prior to the test, only fever was observed as core symptom in the S-based group. We found that falling and somnolence were the most common reported other symptoms prior to the test in the TP-based group. Thus, extra attention should be paid to these symptoms since they might indicate a possible infection. This is in line with previous studies, in which symptoms such as falling and delirium were reported, but in which debuts with core symptoms were more common . In the In our study, 40% of the residents with confirmed COVID-19 died within 30 days. This percentage is higher than the 34% reported by McMichael et al.,  and the This study had some limitations that must be acknowledged when interpreting results. The study was carried out within one organization and based on EHR data. Because of the use of EHR data, we may have missed symptoms and also workload during the COVID-19 pandemic may have influenced symptom registration in the EHR negatively. As we used both care staff and physicians registration, this might have been somewhat overcome. Measuring the oxygen saturation and especially the temperature  will not have been the same in every ward, but it is close to practice. A RT-PCR test has relatively low sensitivity (63–78%) . ConsequMany NH residents in this investigated sample with positive PCR did not have core symptoms when tested  but had other signs/symptoms at the day of testing and in the week before the positive test. Yet, a large part of this group did develop these core symptoms after the testing day. Daily fluctuations in temperature and oxygen saturation can contribute to earlier detection. The results of this study underscore the importance of current testing policies that advice ample and repeated testing of all residents and personnel that are in close contact with a resident with confirmed COVID-19. This study shows that course profiles may be present. However, in order to substantiate this with more certainty, more research is needed that is prospective and longitudinal.


In vivo, PsEUL-OVA could significantly increase the levels of OVA-specific antibody  titers and cytokine  levels. Additionally, it could activate T lymphocytes and facilitate the maturation of dendritic cells (DCs). These findings collectively suggested that PsEUL-OVA induced humoral and cellular immune responses by promoting the phagocytic activity of macrophages and DCs. Taken together, these results revealed that PsEUL-OVA had the potential to improve immune responses and provide a promising theoretical basis for the design of a novel delivery system.In this investigation, to maximize the desired immunoenhancement effects of PsEUL and stimulate an efficient humoral and cellular immune response against an antigen, PsEUL and the model antigen ovalbumin (OVA) were coupled using the N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) reaction to yield a novel delivery system (PsEUL-OVA). The physicochemical characteristics and immune regulation effects of this new system were investigated. We found the yield of this EDC method to be 46.25%. In vitro, PsEUL-OVA (200 μg mL Eucommia ulmoides Oliv. (EU) has been used as traditional Chinese medicine for at least 2000 years and is commonly known as “Du-Zhong” in China. It is obtained from the stem bark of 15- to 20-year-old Eucommia ulmoides Oliv. trees and was recorded in ancient Chinese medical texts as “Shennong’s Herba”. This ancient nourishing herb was commonly used as a tonic to strengthen bones and muscles and nourish the liver and kidneys. This herb was also used as a diuretic and as a treatment for a variety of pathological conditions, including arthritis, recurrent miscarriage, rheumatism, and lumbago [Mw) of Eucommia ulmoides Oliv. Polysaccharides (EUPS) was determined to be 11.4632 × 105 Da. The monosaccharide components of EUPS are glucose, fructose, mannose, fucose, galactose, and arabinose, with a relative mass of 36.6%, 16.6%, 14.2%, 15.7%, 9.5%, and 7.4%, respectively [ lumbago . AccordiEliciting an immune response is the most effective way for the body to fight harmful substances. Currently, naturally occurring polymer antigens, such as proteins, are commonly used , and theNeisseria meningitidis and pneumonia by Streptococcus pneumoniae. These conditions have a common causative factor, i.e., the bacterial capsular polysaccharide [Naturally occurring macromolecules, especially plant polysaccharides, have immunomodulatory effects on the body owing to their unique structural properties ,12,13. Pccharide ,21. Althccharide ,23,24.In general, the use of polysaccharide-binding proteins is one of the ways for these treatments to complement each other, whether they are protein- or polysaccharide-based, as the primary aim is to improve drug delivery. Many researchers have focused their attention on coupling proteins with polysaccharides using the EDC condensation reaction. EDC activates proteins and the activated proteins are chemically bonded to the polysaccharides; these synthetic products can retain the activity of proteins and polysaccharides to the greatest extent while being absorbed by the body to elicit a pharmacological response ,26.In this study, we conjugated PsEUL with OVA for the first time, using the EDC method to synthesize an antigen delivery system (PsEUL-OVA) that uses PsEUL to assist OVA and hypothesized that the specific immune response of antigens could be increased using this method. We evaluated the immune response of PsEUL-OVA in vitro and in vivo. The aim of this investigation was to evaluate the use of the EDC method to conjugate PsEUL and OVA to improve their immune-enhancing activity, thus providing crucial application prospects in drug delivery system development.Eucommia ulmoides Oliver leaf was obtained from SPH Zunyi Pharmaceutical Co., Ltd. . OVA and Freund’s Adjuvant, Complete, was purchased from Sigma-Aldrich Co., LLC . EDC and ADH were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. . Sephadex G-150 was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. . Cell Counting Kit-8 (CCK-8) was purchased from Shanghai Beyotime Biotechnology Co., Ltd. . The mouse macrophage (RAW264.7) cell line was obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. . Phalloidin-iFluor 555 Reagent was obtained from Abcam Inc.  and 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Solarbio . HRP goat anti-mouse IgG, IgG1, IgG2a, and IgG2b were obtained from Santa Cruz Biotechnology, Inc. . IL-2, IL-4, IL-6, and an IFN-γ mouse uncoated enzyme-linked immunosorbent assay (ELISA) kit were obtained from Thermo Fisher Scientific Inc. .E. ulmoides leaf powder was ground and passed through a 60-mesh sieve. Next, the sieved leaf powder was mixed in a ratio of 1:20 with distilled water and was subjected to ultrasonication in a water bath at 60 °C for 2 h. The filtrate was evaporated under reduced pressure and the protein was removed using the Sevag method [PsEUL was obtained via hot water extraction and alcohol precipitation . E. ulmog method  by treatg method .About 800 mg PsEUL was dissolved in 200 mL sodium chloride solution (0.15 M) and reacted with cyanogen bromide at 20 °C for 15 min (pH 10.8). Then, 200 mL of ADH solution was added and the pH of the reaction mixture was adjusted to 8.6 using 0.5 M hydrochloric acid solution; the mixture was left at 20 °C overnight. The next day, the solution was dialyzed (10 kDa) against 20 mM EMS buffer to remove excess ADH and cyanogen bromide. This process is called polysaccharide activation . A volumSeparation and purification of PsEUL-binding OVA (PsEUL-OVA) were performed using column chromatography and the column volume (1.2 × 30 cm) was 300 mL. The sample was loaded on a Sephadex G-150 column equilibrated with PBS. After loading, the sample was eluted using a flow rate of 1 mL/min for the PBS (pH 7.4) solution. The eluent was collected using an automatic collection device  at a rate of one tube per minute using a Biuret kit. The collected fractions were detected at a wavelength of 540 nm and the elution curve was drawn. The separated and purified binding proteins were freeze-dried and the resultant powder was stored at 4 °C .−1 [PsEUL and PsEUL-OVA were measured using Fourier transform infrared spectrometry . Samples (1–2 mg) were mixed with potassium bromide and ground thoroughly. The powder was pressed into sheets using a tablet press and scanned in the band of 4000–400 cm−1 .PsEUL-OVA was identified using SDS-PAGE in a discontinuous buffer system consisting of 5% stacking gel and 12% separating gel. The sample was dissolved in distilled water, SDS-PAGE sample loading buffer (4:1) was added, and each lane was loaded with 10 μL for electrophoresis. After electrophoresis, the gel block was stained using Coomassie Blue staining solution for 1 h and decolorized using Coomassie Blue destaining solution for 2 h. Protein markers ranging from 10–150 kDa were used as standards .Appropriate amounts of PsEUL and PsEUL-OVA were taken and adhered to the sample table using copper tape. The sample table was placed on an ion sputtering instrument, coated with a layer of conductive gold powder, and was placed under the SEM system  and observed. Each sample was photographed three times to eliminate sample interference and system errors .PsEUL, OVA, and PsEUL-OVA were dissolved in distilled water, the solution was added to a quartz cuvette, and the particle size was measured using a laser particle-size analyzer  .PsEUL, OVA, and PsEUL-OVA were dissolved in distilled water, the solution was added to a disposable folded capillary sample cell. The samples were analyzed using a laser particle size analyzer  to measure its zeta potential .PsEUL-OVA was separated using column chromatography and purified. The BCA method  was usedPolysaccharide-binding protein conversion rate (100%) = After adjusting the cell concentration of macrophages, they were placed in a 96-well cell-culture plate. After 24 h of culture, different concentrations of polysaccharide-binding protein solution at 50, 100, 150, 200, 250, 300, and 350 μg/mL were added and cultured for a further 24 h. Next, 10 μL of Cell Counting Kit-8 (CCK-8) reagent was added to each well and the OD was determined using a microplate reader at 450 nm after 4 h. Based on the OD value, the most suitable drug concentration was calculated using the following cell activity formula, as stated in the CCK-8 kit:S: with cells, CCK-8 solution absorbance of drug wells; AB: absorbance of wells with medium and CCK-8 solution without cells; AZ: absorbance of wells with cells and CCK-8 solution without drugs).(AThe PsEUL-OVA was mixed with fluorescein isothiocyanate (FITC) in dimethyl sulfoxide (DMSO) at 4 °C for 12 h, dialyzed against PBS at 4 °C for 72 h, and lyophilized into a powder to obtain FITC-OVA-PsEUL. A similar method was used to prepare FITC-OVA. Macrophages were inoculated in a 6-well cell-culture plate with a round coverslip. After 24 h of culture, FITC-OVA-PsEUL and FITC-OVA were added; after a further incubation of 12 h, the slides were taken out and fixed with 4% paraformaldehyde for 20 min and permeabilized with Triton X-100 for 4 min. Next, they were stained with Phalloidin-iFluor 555 Reagent for 50 min and with DAPI staining solution for 5 min. Lastly, the macrophages were mounted with 90% glycerol and photographed using a confocal laser scanning microscope .Female ICR mice  were provided by Beijing Charles River Laboratory Animal Technology Co., Ltd., . All animal procedures were performed as per internationally accepted principles mentioned in the Guidelines for Keeping Experimental Animals issued by the Government of China. The protocols were approved by the IACUC, Southwest University (NO: IACUC-20191223-16). Ninety 4-week-old female ICR mice were selected and randomly divided into 6 groups of 15 mice each. The first immunization was performed after one week of adaptive feeding. Mice in the blank group were injected subcutaneously with 0.2 mL of saline. The second group comprised the PsEUL group and mice were injected with 0.2 mL (500 μg/mL) PsEUL solution. The third group was the OVA group and mice were injected with 0.2 mL (500 μg/mL) OVA solution. In the PsEUL + OVA group, mice were injected with 0.2 mL PsEUL and 0.2 mL OVA (500 μg/mL) solution. PsEUL-OVA constituted the fifth group and mice were injected with 0.2 mL (1000 μg/mL) PsEUL-OVA solution. The sixth group was a positive control group and mice were injected with 0.2 mL OVA (500 μg/mL) in FCA and a distilled water mixed solution (1:1). A booster immunization was carried out once a week and a total of four immunizations were performed. Each immunization dose was similar to that of the first immunization .One week after the first immunization, 3 mice were randomly selected from each group for blood collection; the serum was separated and stored in a −20 °C refrigerator. The thymus and spleen were collected to calculate the immune organ index. The above procedures were performed every week thereafter throughout the duration of the immunization program. The mouse immune organ index was calculated using the following formula:After 3 and 28 d following the first immunization, 3 mice were randomly selected from each group for flow cytometry. Their spleens were collected and ground to make a single-cell suspension. PE and FITC double staining methods were used; the cell surface staining markers included CD3, CD4, CD8, CD11c, MHC-II, CD80, and CD86. The double-stained cell suspensions were transferred to a flow cytometer tube and analyzed using flow cytometry .2SO4. The OD at 450 nm was measured using a microplate reader .The OVA-specific IgG and IgG subtypes  in serum samples were detected using ELISA. Briefly, the coating solution containing OVA was added to the 96-well ELISA plate for 18 h. After three washes, 5% of skim milk was added to the block and incubated at 37 °C for 1 h. After three washes, horseradish peroxidase (HRP)-labeled goat anti-mouse antibody  was added to each well and incubated for 1 h at 37 °C. Lastly, the serum samples were added to the 96-well plate and incubated at 37 °C for 1 h. After washing, the substrate was developed using 3,3′,5,5′-tetramethylbenzidine (TMB) and then the reaction was terminated by adding 2 M HCytokines IL-2, IL-4, IL-6, and IFN-γ in mouse serum were measured using an ELISA kit and a microplate reader was used to measure the OD at 450 nm.p < 0.05). The order of statistically different activities in letters is as follows a > b > c > d > e.Test results are expressed as the mean ± standard deviation. SPSS 22 software  was used for data analysis. Duncan and LSD multiple range tests were used to determine the differences among groups. Different letters  above each group indicate statistically significant differences . The level of glucose was highest and found to be between 38.2% and 39.1%, followed by L-arabinose 37.7%), D-galactose (12.8%), and L-rhamnose monohydrate (11.8%). Alduronic acid accounted for 21.2% of the important components of PsEUL. The molecular weight of PsEUL ranged from 6 × 10.7%, D-gaSephadex G-150 column chromatography was used to purify PsEUL-OVA ,40. The −1, and the results are shown in −1 [−1, C-H stretching vibration of CH3, CH2, and CH was observed [−1, the stretching vibration of the amide group C=O at 1056 cm−1 and 461 cm-1 indicated the presence of the pyranose ring. At 866 cm−1, the variable angle vibration of CH in the β-pyran ring, at 818 cm−1 was observed, indicating the vibration absorption peak of the C-H variable angle in the furan ring [−1 was lower than that of PsEUL. This finding indicated that the O-H of PsEUL was activated by CNBr and corresponded to the variable-angle vibration absorption peak of NH at 1514 cm−1 [−1 corresponded to the CN stretching vibration in EDC [−1, indicating that the PsEUL had succeeded in coupling to OVA through the formation of a covalent bond (-CO-NH-).FT-IR was used to scan PsEUL and PsEUL-OVA in the band of 4000–400 cmwn in −1 . At 2922observed . At 1654ran ring . Based o514 cm−1 . 1124 cmn in EDC . At the It is well known that the condensation reaction using EDC results in an increase in the molecular weight of the protein. The molecular weight of OVA is 44.5 kDa. Previous studies have reported that the molecular weight of PsEUL is between 60 and 600 kDa; after the coupling of PsEUL and OVA, the molecular weight of PsEUL-OVA would, therefore, be significantly higher than that of OVA. As shown in It can be seen from the SEM images at different magnifications that PsEUL appears as a relatively smooth and dense block at 100 times, whereas a large number of fine particles are attached to the surface of PsEUL-OVA. At a higher magnification, it can be seen that the surface of PsEUL-OVA is smoother than that of PsEUL and there is a clear connection between the particles. In general, SEM images indicate that the structure of PsEUL and PsEUL-OVA has changed significantly. The images also indicate that PsEUL-OVA exhibits the adhesion of objects, thereby depicting the successful combination of OVA and PsEUL C.Previous analysis shows that PsEUL-OVA has a relatively large molecular weight. Studies have confirmed that the size of the drug is related to its systemic bioavailability . TherefoThe conversion rate of polysaccharide-binding protein (%) = 46.25% calculated using the BCA and phenol sulfate methods. The ratio of polysaccharide to protein was determined to be 12:25.−1 and even increased cell proliferation. The maximum cell proliferation activity was observed at a concentration of 200 μg mL−1 and was significantly different from the blank control group (p < 0.05). Therefore, a concentration of 200 μg mL−1 was selected for subsequent experiments.As important immune regulatory cells of the immune system, macrophages not only participate in eliciting specific and non-specific immune responses but also serve as “bridge cells” between the two . TherefoIn order to explore the ability of macrophages to take up PsEUL-OVA, the phagocytic effect of macrophages on antigens was observed using laser confocal scanning microscopy. Different fluorescence intensities indicate the uptake of different antigens by macrophages. It can be seen in p < 0.05). The organ index in the PsEUL-OVA group was significantly higher than that of the FCA+OVA group (p < 0.05). Our results demonstrate that PsEUL-OVA could promote the immune organ index.The thymus and spleen are important immune organs, responsible for lymphocyte production . When thp < 0.05), whereas the PsEUL+OVA group produced specific IgG and no significant difference was observed compared to the OVA group (p > 0.05). Among them, the specific IgG produced by the PsEUL-OVA group was significantly higher than that in the FCA+OVA group on days 7 and 28. There was no significant difference between the specific IgG produced by the PsEUL-OVA and FCA+OVA groups on days14 and 21 (p < 0.05). Our results showed that PsEUL-OVA could induce a high level of OVA-specific IgG and indicated a positive correlation between antibody levels and the number of immunizations.We explored the effect of PsEUL-OVA on the humoral immune response of mice. OVA-specific IgG and IgG subtypes in mouse serum were measured using ELISA on days 7, 14, 21, and 28 after the first immunization. Results of OVA-specific IgG antibodies in mouse serum are shown in p < 0.05). As illustrated in p < 0.05). The IgG2a in the PsEUL-OVA group was significantly higher than that of the FCA+OVA group on days 7, 14, and 21 (p < 0.05) and there was no significant difference between the PsEUL-OVA and FCA+OVA groups on day 28 (p > 0.05). It was observed that the amount of IgG2a produced by mice immunized with PsEUL-OVA was not directly related to the number of immunizations. The levels of IgG2b in the PsEUL-OVA and FCA+OVA groups were significantly higher than that in the OVA and PsEUL groups (p < 0.05). The IgG2b antibody titer produced by the PsEUL-OVA group was significantly higher than that of the FCA+OVA group (p < 0.05), whereas the amount of IgG2b produced by the PsEUL-OVA group on day 21 was highest. This finding suggested that PsEUL-OVA could dramatically upregulate the OVA-specific subtype antibody titers.OVA-specific subtypes in mouse serum are illustrated in As shown in p < 0.05). The IL-4 and IL-6 concentrations in mice, especially in the PsEUL-OVA group were significantly higher than those in the blank and OVA groups  cells. CD4+ and CD8+ T cells, using labeled CD3+ cells, can filter out mature CD4+ T and CD8+ T lymphocytes, respectively. The ratio of CD4/CD8 reflects the body’s immune function [+ T lymphocytes in the PsEUL-OVA group is significantly higher than that in the FCA+OVA, PsEUL+OVA, and naive groups (p < 0.01). The levels of CD8+ T lymphocytes of mice in the PsEUL-OVA group were significantly higher than those of mice in the other groups (p < 0.01). The CD4/CD8 ratio in the PsEUL-OVA group was significantly higher than that in the other groups (p < 0.01). Data from the present study suggested that PsEUL-OVA could induce a stronger cellular immune response in immunized mice and also demonstrated that PsEUL could function as an adjuvant conjugated with OVA and induce a Th1-biased immune response in mice.CD3function ,55,56. I+, CD86+, and MHCⅡ in dendritic cells [+ in the PsEUL-OVA group was significantly higher than that in the FCA+OVA, PsEUL+OVA, and naive groups (p < 0.01). The expression level of CD86+ in the PsEUL-OVA group was significantly higher than that in all other groups . Results from the present study indicated that PsEUL-OVA could increase the maturation of DCs.The maturation of dendritic cells can reflect the level of cellular immune response and can be evaluated by determining the expression levels of CD80ic cells . As illu+ and CD8+ on spleen lymphocytes, and maintained the CD4+/CD8+ balance. Moreover, PsEUL-OVA stimulated Th1/Th2 cytokine and antigen-specific IgG and IgG subclass antibody production. Our results reveal that PsEUL-OVA efficiently delivered the OVA to DCs and significantly promoted antigen-presenting efficiency.In recent years, polysaccharide-conjugated delivery systems have gained much attention in the field of applied materials science, and several studies have shown that polysaccharide modifications can greatly increase antigens’ immune responses ,31. In tThe EDC condensation reaction is a widely used method that can activate proteins. When chemically bonded to polysaccharides, synthetic products can retain the activity of proteins and polysaccharides to the greatest extent when absorbed by the body ,26. In t−1, the activity of macrophages was found to be highest; therefore, this concentration was used for experiments related to cell phagocytosis. Results concerning the phagocytic efficiency of macrophages against antigens, observed using laser confocal scanning microscopy, revealed the improved ability of macrophages to phagocytose antigens in the presence of PsEUL. After coupling the antigen with PsEUL, macrophages could take up more antigens, likely due to specific polysaccharide receptors on the macrophage surface. A large amount of PsEUL-OVA was adsorbed on the cell surface of macrophages, which greatly increased the ability of macrophages to take up the antigen.Macrophages are monocytes with strong phagocytic ability, especially against pathogens . MacrophImmunoglobulin (Ig) is a protein secreted by effector B cells that can specifically bind to the corresponding antigen when the antigen stimulates the body’s immune system. Among these, IgG is the most common, and most antibody reactions involve IgG-mediated effector functions. The presence of specific IgG and its subclasses in serum reflects the body’s immune level and the expression level of different subclasses of IgG determine the type of immune response . The proCytokines are mainly small molecule polypeptides or glycoproteins synthesized and secreted by immune cells. Cytokines can mediate the interaction between cells and regulate the immune response, including the production of interleukins (IL), interferons (IFN), and tumor necrosis factor (TNF) ,61,62. THuang et al. confirmed that the Arabinogalactan-poly (I:C) (AG-P) was conjugated with Ag85B-HspX fusion protein (AH) to generate a vaccine (AH-AG-P) that can induce cytokine  production in C57BL/6 mice . In the + T cells and CD8+ T cell subsets. CD4+ T cells are important immune cells in the body’s immune system. CD4 is a T cell antigen receptor (TCR) that activates CD4+ T cells to secrete many different cytokines to regulate the function of cytotoxic T lymphocytes (CTL) and B cells. CD8 is also a Th cell TCR; CD8+ T cells differentiate into CTL after activation and can specifically kill target cells. Therefore, the CD4+ T/CD8+ T ratio is used clinically to reflect the activity of T lymphocytes [+ T cells in C57BL/6 mice [+CD4+ and CD3+CD8+ double-positive cells and found that the number of CD4+/CD8+ T cells in the PsEUL-OVA group mice increased significantly, indicating that CD4+ T cells could proliferate after immunization with the PsEUL-OVA and regulate CTL activity.T lymphocytes can be divided into CD4phocytes . Huang eL/6 mice . In the DCs are currently the most powerful immune cells for antigen presentation. Immature DCs are induced into mature DCs under the action of co-stimulatory signaling molecules. CD80 is an important substance for T lymphocyte activation. Although CD86 can induce T lymphocytes to produce IL-2, MHCⅡ is a sign of the maturation of DCs . In thisTo date, vaccination is the most effective way to control infectious diseases. The antigen delivery systems or adjuvants are required to promote immune response production or increase the effectiveness of vaccines . Some naIn summary, we have demonstrated the feasibility of an antigen delivery system, PsEUL-OVA, in delivering OVA to macrophages and DCs. PsEUL-OVA significantly induced cellular and humoral immune responses by facilitating DC maturation. This PsEUL-based delivery system showed strong immunomodulation efficiency in promoting antigen presentation and activating T cells, and it could be applied to improve the effectiveness of new vaccines. However, the mechanism of this modulation effect of the PsEUL-based delivery system needs to be further investigated. This study has created a platform for future research studies on adjuvant and antigen delivery and the prolongation of their efficacy.Herein, for the first time in the field, we explored the chemical coupling of PsEUL and OVA to yield PsEUL-OVA. Our results indicated that PsEUL-OVA has good immune-adjuvant activity and could enhance immunogenicity. In vitro, PsEUL-OVA not only promoted the proliferation of macrophages but also enhanced the phagocytosis of macrophages. PsEUL-OVA could not only activate a stronger antigen-specific immune response and enhance cytokine levels in vivo, but also exhibited a Th1-type biased immune response that prompted the maturation of T lymphocytes and DCs. Taken together, the results of our study help us to understand the positive regulation of PsEUL-OVA in the immune system. These findings pave the way towards using PsEUL-based drug delivery systems for future vaccine formulations and delivering nanotherapeutics to diseased cells, improving their potency and efficacy. Future studies will further investigate the underlying mechanisms of the PsEUL-based drug delivery system in relatin to the immune response, and its potential application in other animal models. Our study shows the prospect of developing natural polysaccharide-conjugated antigen nanodelivery systems to improve the body’s immune response.


Objective: To validate an Arabic version of the Danish Prostatic Symptom Score (DAN-PSS), a self-administered quality-of-life questionnaire.Patients and methods: The reliability of the Arabic DAN-PSS was assessed by determining the internal consistency (Cronbach’s α coefficient) and by assessing the test–retest reliability (Kappa [κ] test). Inter-domain associations were examined using Spearman’s correlation coefficient (r). The discrimination validity was evaluated using receiver operating characteristic (ROC) curves. The sensitivity to change of the questionnaire and its individual items was assessed before and after intervention using a paired t-test.Results: In all, 106 men (55 patients with BPH and 51 without BPH symptoms) were included. A high level of internal consistency amongst the three domains of the answered Arabic DAN-PSS questionnaire was observed (Cronbach’s α > 0.70). Also, there was a good correlation between storage and voiding  and post-micturition symptoms domains . Voiding and post-micturition symptoms domains also had a good correlation . The agreement between the test and retest scores had a κ value of 0.83 (P < 0.001). The ROC curve had an area under the curve of 0.98. The sensitivity to change comparing patients with BPH who received medical or surgical intervention revealed Arabic DAN-PSS mean (SD) scores of 34.7 (17.7) and 17 (8.7) before and after the intervention, respectively (P < 0.001).Conclusion: The Arabic DAN-PSS is a clear questionnaire, valid, reliable, and responsive that can be used for BPH associated with lower urinary tract symptoms assessment and follow-up in clinical practice and research in Arabic-speaking patients.Abbreviations: AUC: area under the curve; BPH: benign prostatic hyperplasia; CI: confidence interval; DAN-PSS: Danish Prostatic Symptom Score; DRE: digital rectal examination; ICIQ-MLUTS: International Consultation on Incontinence Male LUTS Questionnaire; ICS: international continence society; IPSS: international Prostatic Symptom Score; IPSS-Arb: Arabic version of the IPSS; LUTS: lower urinary tract symptoms; PSA: prostatic specific antigen; PSS: prostatic symptom score; QoL: quality of life; ROC: receiver operating characteristic; UTI: urinary tract infection. BPH associated with LUTS (BPH/LUTS) affects >20%  of men aged 30–79 years, reaching 80% of men by the age of 70 years, and causes bother and impairs quality of life (QoL) . The ICSThe European Association of Urology (EAU) guidelines for male LUTS/BPH  recommenSymptom scores are supportive in quantifying and identifying which symptoms are predominant. These validated symptom scores are sensitive to symptom changes and can be used to monitor treatment . Arabic We thought that a validated Arabic version of the DAN-PSS would help assess prostate-related symptoms and QoL to improve patient management as a supportive tool in national and international research trials. Thus, the present study aimed to develop an Arabic version of the DAN-PSS and to assess its validity and reliability.This prospective study of the development of the Arabic DAN-PSS was followed by an assessment of the validity and reliability of this Arabic version between May 2019 and August 2020 at a tertiary teaching hospital after approval by the Institutional Review Board. Patients complaining of BPH/LUTS, seeking medical advice and who could read were invited to participate in our trial. Exclusion criteria were neurological bladder, diabetes mellitus, or acute UTI. BPH was diagnosed clinically based on history, including the validated Arabic version of the IPSS (IPSS-Arb) and physical examination, including a DRE, serum PSA levels, uroflowmetry, and pelvi-abdominal ultrasonography.The English DAN-PSS was initially translated into Arabic. Translation to the cultural context and lifestyle related to the Arabic language was performed by a bilingual native Arabic speaker having experience of the language translation of clinical symptom questionnaires . This Arabic version was back-translated. Back-translation from Arabic into English was carried out .Review of the translation and back-translation by a panel of native Arabic and English speakers, respectively, to identify any meaning errors and discrepancies. Any differences between the versions were resolved through a consensus meeting between the translator and the urologists . This rePre-testing was performed using a sample of 20 patients, who represented a sample of potential respondents to the questionnaire, to assess its comprehensiveness. They reported no problems in completing and answering the questions clearly and simply. Those 20 patients were excluded from the final analysis.et al. [Based on the Hansen et al.  and the et al. , for estr). A good correlation was considered when r was >0.5. The Arabic DAN-PSS stability was assessed by a 2-week test–retest analysis using the weighted Kappa (κ) test. The discrimination validity was evaluated by comparing the scores of cases with those of controls using receiver operating characteristic (ROC) curves [t-test to compare the scores difference before and after the BPH treatments. All statistical tests were done using the Statistical Package for the Social Sciences (SPSS®), version 21 ; with a P < 0.05 considered statistically significant.The reliability of the Arabic DAN-PSS was evaluated for internal consistency using Cronbach’s α for each domain. A Cronbach’s α value of ≥0.70 was considered acceptable for internal consistency . Domain ) curves  and MannIn all, 106 men  were included in the assessment of the validity of the Arabic DAN-PSS. The mean (SD) age was 61 (7.8) and 28 (6.1) years in BPH and normal controls, respectively. With regards to the level of education, 57 (53.8%) of the participants held a university degree, 31 (29.2%) had graduated from high school, and 18 (17.0%) had completed primary education. Regarding the BPH group, the mean IPSS was 9.1, with a corresponding 95% CI of 7.7–10.6. The mean (95% CI) post-void residual urine volume was 60 (20–100) mL. The mean (95% CI) serum PSA level was 4.8 (2.5–7.1) ng/mL.r = 0.75; P < 0.001) and post-micturition symptoms domains . The voiding and post-micturition symptoms domains also had a good correlation .In the BPH group, we found a high level of internal consistency amongst the three domains of the answered Arabic DAN-PSS questionnaire with Cronbach’s α > 0.70 . Also, tP < 0.001). The ROC curve constructed from the Arabic DAN-PSS total scores had an AUC of 0.98, which means that the Arabic DAN-PSS was able to discriminate between patients with BPH and control subjects who had no voiding problems in general 98% of the time. Using the ROC curve, a total Arabic DAN-PSS of ≥5 was the cut-off value associated with a sensitivity of 98% and specificity of 86% (P < 0.001) for prediction of patients with LUTS/BPH (P < 0.001). The sensitivity to change of the Arabic DAN-PSS was assessed in 26 patients with BPH who received medical or surgical intervention. The mean (SD) scores were 34.7 (17.7) and 17 (8.7) before and after the intervention, respectively (P < 0.001).The agreement between the test and retest scores had a κ value of 0.83 (LUTS/BPH . There wIn men aged ≥40 years LUTS are highly prevalent and often bothersome symptoms, which have a major impact on health-related QoL, mental health, and workplace productivity that can result in economic effects ,12. SeveLUTS are divided into three groups: storage, voiding, and post-micturition symptoms . As a reet al. [The DAN-PSS developed by Hansen et al.  includeset al. ,12.The reliability of the Arabic version of the DAN-PSS, which refers to its ability to measure reproducibly  was confThe discriminant validity of the Arabic DAN-PSS revealed an area under the ROC curve  equal 0.The limitations of our present trial are that the DAN-PSS used the standard Arabic language and tested it in a single hospital. It will need to be tested in different Arabic countries and communities, as the dialects differ from one country to another. We had some selection bias by excluding illiterate patients whom would need someone else to read and answer the questionnaire for them. Also, there was an age difference between the control and BPH/LUTS groups.The Arabic DAN-PSS is a clear questionnaire, valid, reliable, and responsive that can be used for BPH/LUTS assessment and follow-up in clinical practice and research in Arabic-speaking patients.


Background: Dianbaizhu (Gaultheria leucocarpa var. yunnanensis), a traditional Chinese/ethnic medicine (TC/EM), has been used to treat rheumatoid arthritis (RA) for a long time. The anti–rheumatic arthritis fraction (ARF) of G. yunnanensis has significant anti-inflammatory and analgesic activities and is mainly composed of methyl salicylate glycosides, flavonoids, organic acids, and others. The effective ingredients and rudimentary mechanism of ARF remedying RA have not been elucidated to date.Purpose: The aim of the present study is to give an insight into the effective components and mechanisms of Dianbaizhu in ameliorating RA, based on the estimation of the absorption, distribution, metabolism, and excretion (ADME) properties, analysis of network pharmacology, and in vivo and in vitro validations.Study design and methods: The IL-1β–induced human fibroblast-like synoviocytes of RA (HFLS-RA) model and adjuvant-induced arthritis in the rat model were adopted to assess the anti-RA effect of ARF. The components in ARF were identified by using UHPLC-LTQ-Orbitrap-MSn. The quantitative structure–activity relationship (QSAR) models were developed by using five machine learning algorithms, alone or in combination with genetic algorithms for predicting the ADME properties of ARF. The molecular networks and pathways presumably referring to the therapy of ARF on RA were yielded by using common databases and visible software, and the experimental validations of the key targets conducted in vitro.Results: ARF effectively relieved RA in vivo and in vitro. The five optimized QSAR models that were developed showed robustness and predictive ability. The characterized 48 components in ARF had good biological potency. Four key signaling pathways were obtained, which were related to both cytokine signaling and cell immune response. ARF suppressed IL-1β–induced expression of EGFR, MMP 9, IL2, MAPK14, and KDR in the HFLS-RA .Conclusions: ARF has good druggability and high exploitation potential. Methyl salicylate glycosides and flavonoids play essential roles in attuning RA. ARF may partially attenuate RA by regulating the expression of multi-targets in the inflammation–immune system. These provide valuable information to rationalize ARF and other TC/EMs in the treatment of RA. Rheumatoid arthritis (RA) is a chronic autoimmune disease with a higher prevalence in women, which according to Gaultheria leucocarpa var. yunnanensis (Franch.), known as “Dianbaizhu” in TC/EMs, belongs to the Ericaceae family and is mainly distributed in Southwest China. It has been widely used as a folk medicine for the treatment of inflammatory diseases, such as RA and chronic tracheitis in the Yi nationality ; 2) the organ indexes (OIs) in adjuvant-induced arthritis (AIA) in rats were measured in vivo, histological and pathological changes of the joints were surveyed, and the repression capability of ARF on RA, as well as the release of inflammatory factors, was analyzed; 3) QSAR models of the ADME features based on using RF, SVM, KNN, PLS-DA, and EBPT alone or in combination with the GA were proposed. The ADME-related parameters of the compounds of ARF were predicted and evaluated; 4) protein–protein interaction (PPI) network analyses, the KEGG pathway, and gene ontology (GO) enrichment analyses were performed for key targets; 5) the compound–common target and RA-related pathway networks were developed to investigate the potential mechanisms, and the key targets were selected for subsequent experimental verification in vitro. Consequently, a better comprehension of the underlying pharmacodynamics of ARF could provide new insights for treatment against RA. At present, this exploration strategy could present the critical active ingredients and potential mechanisms of ARF in relieving RA and bring weighty benefits in screening new clinical therapeutic approaches on RA. The whole framework diagram of this study is depicted in A systematic study of the multiple components and multiscale mechanisms was carried out to investigate the remedial effect of ARF on RA, and the following steps were taken: 1) the components of ARF of Dianbaizhu was collected from Chuxiong . Methotrexate (MTX) was bought from SPH Sine Pharmaceutical Laboratories Co. Ltd. . Aspirin was purchased from Jiangsu Pingguang Pharmaceutical Co. Ltd. . Freund’s complete adjuvant  and β–actin (A5441) were obtained from Sigma (USA). Human fibroblast-like synoviocytes of RA (HFLS-RA) were purchased from Hunan Fenghui Biotechnology Co. Ltd. . The DMEM/F12 medium (18219003), FBS (190913), and penicillin streptomycin solution (30002303) were obtained from Beijing BioDee Biotechnology Co. Ltd. . IL-1β (P01584) was supplied by Novoprotein Technology Co. Ltd. . Rat IL-2 ELISA kit (KA30281115), Rat IL-1β ELISA kit (KA301B90421), Rat IL-6 ELISA kit (KA30690741) and Rat TNF-α ELISA kit (KA38290222) were bought from Beijing Biodragon Immunotechnologies Co., Ltd. . The primary antibody (anti-rabbit) and secondary antibody (anti-mouse) were bought from Cell Signaling Technology, Inc. . Antibodies against the following proteins were used: EGFR (18986-1-AP), MMP9 (10375-2-AP), IL2 , MAPK14  and KDR . These antibodies as well as RIPA Lysis Buffer (S1004), protease inhibitor (KGP603), BCA KIT  were provided from Beijing Bioway Biotechnology Co., Ltd. . The AB-8 resin was obtained from Cangzhou Bon Adsorber Technology Co., Ltd. .Gaultheria leucocarpa var. yunnanensis (Franch.) TZ Hsu and RC. Fang from Gaultheria Kalm ex L. (Ericaceae) (G. yunnanensis (17 kg) were extracted using 238-L 30% ethanol–water three times for 2 h each time by the thermal recycling extract method. The solvent was completely evaporated from the ethanol extraction solution to yield a crude extract (approximate 1.7 kg). The total ethanol extract was dissolved in water, and then adsorbed by the AB-8 resin column. It was eluted with distilled water and 35% ethanol–water in order, yielding the 35% elution fraction (approximate 500 g). This fraction was concentrated under reduced pressure to yield ARF powder. After which it was dissolved in distilled water and passed through a 0.22-μm filter for UHPLC-LTQ-Orbitrap-MSn analyses.Dianbaizhu was acquired from the dried aerial parts of icaceae) . Samplesicaceae) . Brieflyn experiment, most of the analysis conditions were consistent with the reported research of For the UPLC-ESI-MSThe operations were performed on all the animals in accordance with the China Physiological Society's Guiding Principles in the Care and Use of Animals, as well as the authorization of the Animal Care Committee of Beijing University of Chinese Medicine.The 8-week-old SPF Wistar rats with a mean weight of (200 ± 20) g, equal numbers of male and female rats, were obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd. (certification number SCXK (Jing) 2016-0006). The animals were housed in suitable temperature and humidity conditions with a 12-h light/dark cycle at the Beijing University of Chinese Medicine. All Wistar rats were allowed to acclimatize themselves for 7 days, and had free access to tap water and food.in vivo. The lowest dosage selection for ARF was twice the daily dose of Dianbaizhu followed for patients with RA (25 g/60 kg body weight). After acclimation for 1 week, the Wistar rats were randomly divided into seven groups (n = 8): the control group (Blank), model group (Model), ARF-treated group 1 , ARF-treated group 2 , ARF-treated group 3 , MTX-treated group (0.5 mg/kg/d), and Aspirin-treated group (100 mg/kg/d). The RA model was established with subcutaneous injections of FCA in the right hind foot. After 7 days of the injection, the ARF-, MTX- and Aspirin-treated groups, respectively, were orally administered ARF, MTX, and aspirin for a period of 21 days. The Blank and Model groups were treated to distilled water.ARF was diluted with distilled water for tests The rats were observed once every two days after primary immunization. The severity of arthritis was evaluated as in previous studies , includiRats were sacrificed by cervical dislocation on the 28th day after the first immunization. Both hind limbs, including the paws and ankles, were dissected, fixed immediately in 4% paraformaldehyde, and embedded in paraffin. Tissue sections (5 μm) were mounted on common slides for staining with hematoxylin and eosin (H&E) or Safranin-O–Fast Green. The histopathological characteristics were evaluated blindly. The data were expressed as mean inflammation scores, and all the sections randomized and evaluated by two trained observers who were blinded to the treatment groups and the severity of arthritis of each rat .The blood samples from all the AIA rats were obtained at day 27 and were anticoagulated with sodium heparin. The levels of IL-1β, TNF-α, IL-6, and IL-2 in the plasma were detected using commercially available rat ELISA kits according to the protocol of the kit, and absorbance was determined at 405 nm .The ADME properties contribute on vital task in early stages of drug discovery process. To evaluate the biological activity of ARF, KNN, SVM, RF, PLS-DA, EBPT, and GA were used to establish the QSAR prediction model for assessing the pharmacokinetic properties, namely oral bioavailability (OB), P-glycoprotein substrate (PGPS), P-glycoprotein inhibitor (PGPI), human intestinal absorption (HIA), and Caco-2 cell permeability (Caco-2).The methods of ML were applied to construct QSAR models according to the references previous reported with some changes . Brieflyn were selected for the next protocol. The active compounds were screened according to the results of the ADME properties prediction by the QSAR models. The potential targets of these compounds were identified with a little change following the protocol published by http://www.lilab-ecust.cn/pharmmapper/, Updated: 2019-04-10) was unitized to predict potential targets of ARF ingredients. In addition, the targets were retained if the fit score was ≥4.0 . The other parameters that were set are Generate Conformers: Yes; Maximum Generated Conformations: 300; Select Targets Set: Human Protein Targets Only; Number of Reserved Matched Targets : 300 (The compounds characterized by using UHPLC-LTQ-Orbitrap-MS00): 300 .With a keyword “rheumatoid arthritis,” the known RA-related targets were obtained mainly from common existing resources, such as DrugBank and OMIM database. Only those drug–target interactions where the drugs were FDA approved for treatment of RA and those targets that were human genes/proteins were used . In addihttps://string-db.org/), and then visualized using the Cytoscape software (The following networks were constructed using Cytoscape software (version 3.6.2): a putative target network of ARF; known RA-related targets network; common target PPI network; compound–target–pathway network of ARF . The PPIsoftware .To assess the topological properties of each node in the networks, four features, namely, the degree value (DV), node degree distribution (NDD), node betweenness centrality (NBC), and node closeness centrality (NCC) were evaluated according to the previous studies . The imphttps://david.ncifcrf.gov)  . As descp-values . Statist2 at 37°C , 1 × 10−3 μM (MTX-2), 5 × 10−4 μM (MTX-3), 5 × 10−5 μM (MTX-4), 5 × 10−6 μM (MTX-5), 1 × 10−6 μM (MTX-6) to HFLS-RA was also investigated. Experiments were performed at 12 h after HFLS-RA seeding. HFLS-RA (6.6 × 104 cells/dish) were incubated with 1 ng/ml IL-1β and at different concentrations of ARF  or MTX (1 × 10−5 μM) for 48 h. To estimate cell proliferation capability, the culture media from the cells exposed to the different concentrations of ARF/MTX, with or without IL-1β, were detected by using the MTT assay  membrane, and sealed with skimmed milk (5%) (Western blotting was conducted following the methods described by ilk (5%) . Secondlilk (5%) . The memt-test or one-way analysis of variance at a significance level of p < 0.05 . This indicates that MTX was harmful to these viscera, while ARF was safe for treating AIA rats. These results show that ARF had reduced the AI score, inhibited synovial hyperplasia, ameliorated cartilage damage, and protected the internal organs of the model rats, yet apparently had not depended on the dosage used.The FCA-induced AIA rats were used in this study to investigate the therapeutic effect of ARF on RA. The morphological characteristics of arthrophlogosis, such as fester and swelling, could be markedly observed in the Model group. However, the treatment groups obviously relieved arthritis severity in the joints of AIA rats . Histopap > 0.05, p > 0.05). The oral administration with high and middle dosages of ARF dramatically repressed the levels of IL-1β and IL-6 in the AIA rats (p < 0.01) compared with those of the Model group (p < 0.01)  . Across Considering prediction accuracy, the number of variables, operation time, model stability, and other factors, the optimized five-property prediction models were finally obtained, which were recorded as PGPI-SVM, PGPS-RF + GA, Caco 2-SVM, HIA-SVM, and OB-PLS-DA + GA, respectively. The detailed information in screening and optimization of the QSAR models of the five properties has been referred to in the results provided in https://www.cnki.net) and PubMed database . The validation results of some compounds are listed in The MDs of 47 components in ARF were calculated according to the PaDEL-Descriptor software. About 703 MD variables were excluded by the MATLAB software according to the pretreatment method described in the A total of 44 overlaps of 260 targets were noted for RA and 303 targets for ARF , indicatThe network centralization and heterogeneity were 0.476 and 1.222, respectively, suggesting that the contribution of some nodes for this network was bigger than that of the others. It was reckoned that these nodes may be pivotal and merit further investigation. As shown in −2), and node closeness centrality , were regarded as the main nodes. A total of 10 targets, including EGFR, MMP 9, IL-2, Mapk14, KDR, and the other five proteins  and Rap1 Signaling pathway , PI3K-Akt signaling pathway  and Ras signaling pathway . These results suggested that ARF may act on these signaling pathways to achieve the treatment purpose for RA.Cytoscape 3.6.2 software was used to draw the component-key target-pathway network diagram of ARF, as shown in ened out . The keyHelicobacter pylori infection pathway and the Ras signaling pathway contained three target genes each. Two target genes were present in other pathways such as for bladder cancer. Particularly, according to the above-mentioned network pathway analyses and published literature, it was found that the critical core proteins or pathways are related to cell physiological processes or inflammation . There is one item related to molecular function, which is the receptor signaling protein tyrosine kinase activity . Taken together, it is speculated that the anti-RA effect of ARF is mainly bound with the biological processes of protein phosphorylation and the activity of receptor signal protein tyrosine kinase.The GO analysis function of the DAVID V6.8 database was used to carry out enrichment analysis on 10 core targets for the anti-RA effect of ARF of Dianbaizhu. About 63 GO items  were obtained , and thep < 0.001, 0.01, 0.05, and 0.05, respectively), indicating that IL-1β can induce HFLS-RA proliferation. In this present study, IL-1β (1 ng/ml) incubation with the cells for 48 h was selected to finally induce inflammation. The potential cytotoxic effects of ARF on HFLS-RA in vitro were tested for safety and effectivity. As shown in −6 to 5 × 10−3 μM) had no remarkable inhibition capability on cell viability of HFLS-RA. ARF (0.0519–1.6626 mg/ml) exhibited prominent suppression on IL-1β–induced hyperplasia of HFLS-RA  on the proliferation of HFLS-RA was first investigated with cell viability as the index. As shown in p < 0.05). The expression of IL-2, MAPK14, and KDR in ARF-L, ARF-M, ARF-H, and MTX groups was decreased (p < 0.05). The expression levels of MMP9 in ARF-H and MTX groups were significantly downregulated (p < 0.05). More interestingly, compared with that of the Blank group, the expression level of EGFR in IL-1β–induced HFLS-RA was reduced without significance (p > 0.05). The expression level of EGFR in ARF-H and MTX group was upregulated after operation, and the expression level was still less than that found for the Blank group (p > 0.05). The expression content of EGFR in the ARF-M and ARF-L groups was not obviously upregulated compared with that in the ARF-H group (p > 0.05). The data shows that ARF could effectively downregulate the content of MMP9, IL2, MAPK14, and KDR in HFLS-RA, prompting that ARF may alleviate the symptoms of RA by targeting MMP9/IL2/MAPK14/KDR proteins.According to the order of DV, NBC, and NCC values in Currently, the main therapeutic approaches for RA are basically focused on relieving symptoms and changing the way of life. Over the past two decades, from relying on broadly active traditional medications, the focus has now shifted toward more specific agents that often target a single receptor, cytokine, or cell type, using monoclonal antibodies, fusion proteins, or targeted small molecules . This chn in the negative ion mode. A total of 48 compounds were characterized in ARF. Subsequently, according to the ADME properties data of known compounds, the QSAR models of ADME properties prediction or classification were established by a series of statistical methods. The ADME parameters of these ingredients in ARF were evaluated using the QSAR models. It has been indicated that ARF had eminent potential biological and pharmacological activities, evincing an excellent drug likeness. In the modeling process, it should be noted that most of the models based on PCA total data set had a long calculation time and poor quality. Therefore, the data matrixes, named PCA-80, were adopted to research on training and modeling by each algorithm. Thus, a total of 50 ADME properties classification prediction models with reliable and correct statistics have been developed. In addition, the simulation results of practical examples have testified that compared with the five single classifiers, the method of the five single classifiers trained by the GA can improve the calculation accuracy and the speed of the convergence process of the models. The results also confirm that the type of properties and single classifiers both have a certain influence on improving the scalability of the GA for the models. Regrettably, among these properties and single classifiers, only RF combined with the GA was more effective in classifying the PGPS of compounds, which plays an important role in QSAR model establishment. The appropriate selection of descriptors by the GA is helpful to the scalability of the models. Too many variables in the model not only have a large amount of calculation, but also may lead to the instability of the side model. In order to solve this problem, variable screening should be carried out. The GA can deal with any form of objective function and constraint, whether linear or nonlinear, discrete or continuous (The disassemble research framework of this study and the relationship between them, as well as the crucial results, were shown in ntinuous . It is sin vitro of ARF on RA through network pharmacology prediction were explored. It is worth mentioning that rutin and isoquercitrin can be detected in ARF incubated by simulated gastrointestinal liquid and human intestinal bacteria in vitro (in vivo experiment, that is, ARF can play an anti-RA role by downregulating the contents of IL-6, IL-1, IL-2, and TNF-α in the plasma of AIA model rats. The results suggest that ARF can affect the occurrence and development of RA by regulating the expression of these five targets, and thereby play a therapeutic role.The underlying action mechanisms and experimental verification in vitro . They arin vitro . Therefoin vitro . The corMoreover, there are few KEGG pathways enriched by the core target/gene of ARF treatment on RA, mainly including proteoglycans in cancer, the Rap1 signaling pathway, the PI3K-Akt signaling pathway, pathways in cancer, the RAS signaling pathway, and so on . It has n. The 50 QSAR models on the ADME properties of these constituents were established on the basis of the ML algorithms. The final optimized model of the five properties was successfully applied to predict the biological activity of ARF. The results showed that the GA was not beneficial to every ML in building and screening the QSAR models, and its advantages depend more on the properties of the components alone. It also needs to be further assessed by extending the number of compounds in the future. The network pharmacology analyses of ARF suggests that methyl salicylate glycosides and flavonoids in ARF are more related to treatment of RA (Gaultheria leucocarpa var. yunnanensis is a folk medicine that the research group of this study have been developing in the past decade, but its pharmacodynamics constituents and mechanism have not been excavated. In the present study, the anti-RA compounds as well as their potential mechanisms were defined, which can be potentially used as a therapeutic option in the treatment of RA without side effects.In this work, 48 major constituents of ARF were characterized by using UHPLC-LTQ-Orbitrap-MSnt of RA . FurtherG. yunnanensis have not been fully tested and verified, which will be further investigated in a follow-up research of this study.For the first time, this investigation offered an integrative analysis by combining drug target and ADME properties prognosis with network analyses to expound the pharmacological mechanisms of ARF in relieving RA. This systematic research is helpful in revealing the rule of TC/EMs of this complex disease of unknown etiology. Owing to the approaching graduation time and unexpected situation with COVID-19, the corresponding ADME properties predicted by the QSAR models in this study, together with the related key targets and pathways predicted by network pharmacology on the anti-RA effect of


Mycobacterium leprae infection can alter the proportions of B cell subpopulations (increase of mature and decrease of memory B cells) in patients affected by leprosy. This modulation is associated with an increase in total IgG and the patient’s clinical condition. Circulating B cells may be acting in the modulation of the immune response in patients with various forms of leprosy, which may reflect the patient’s ability to respond to M. leprae.Despite being treatable, leprosy still represents a major public health problem, and many mechanisms that drive leprosy immunopathogenesis still need to be elucidated. B cells play important roles in immune defense, being classified in different subgroups that present distinct roles in the immune response. Here, the profile of B cell subpopulations in peripheral blood of patients with paucibacillary (TT/BT), multibacillary (LL/BL) and erythema nodosum leprosum was analyzed. B cell subpopulations  and levels of IgG were analyzed by flow cytometry and ELISA, respectively. It was observed that  Mycobacterium leprae, which is endemic in many countries: in 2019, WHO reported 202,185 new cases worldwide  or non-specific pattern recognition receptors. The main mechanisms leading to antibody production by B cells are largely known and, Toll-like receptor (TLR) stimulation in B cells are associated with the regulation of the magnitude of the antibody response and the amount of antigen required for initiating BCR signaling  not only in reactional episodes, but in the pathogenesis of the disease. There are only a few publications about phenotypic analysis of peripheral B cells, restricted to some clinical presentations: Negera et al. studied the total count and frequencies of naïve, mature, and memory  B cells in patients with ENL . Other aPatients with leprosy were recruited from Souza Araújo Leprosy Outpatient Unit  from 2016 through 2019. Uninfected subjects, all residents in the city of Rio de Janeiro , were selected according to the similarity of age (18 to 65) and gender patient’s cohort. The patients were classified on the leprosy spectrum clinically and histologically based on Ridley-Jopling classification schemes . The preThe use of samples was approved by the FIOCRUZ Research Ethics Committee (CAAE 01247418.8.0000.5248). All participants, including parents of minors, provided informed written consent.g for 30 min, then stained with surface antibodies anti-human -CD3 ; -CD19 ; -CD38 ; -CD24 ; -CD27 ; -CD21 ; -IgM  and- IgD  for 30 min at 4°C in the dark. Cells were fixed with 2% paraformaldehyde and stored at 4°C. Data were collected using FACSAria IIup (BD Biosciences) and analyzed using FlowJo software (BD Biosciences)  and mononuclear cells were isolated by centrifugation at 900 x ciences) .The IgG levels were determined in all plasma samples using an in-house ELISA. Briefly, MaxiSorp 96-well-plates  were coated with PBS containing 1.5 μg/ml of anti-human IgG . After overnight incubation at 4°C, the plates were then washed three times with phosphate-buffered saline-0.05% Tween 20 (PBS-Tween) and blocked for 1 h at 37°C with PBS-Tween containing 5% nonfat-dried milk (PBS-Tween-M). Plasma samples diluted 1:1000 in PBS-Tween-M were added in duplicate wells containing 5% nonfat-dried milk (PBS-Tween-M) were added in duplicate wells. After 1 h at 37°C and three washings, specific antibodies were detected by goat peroxidase-conjugated anti-human IgG  and followed by the addition of 3,3′,5,5′-tetramethylbenzidine (TMB) for 30 minutes. The reaction was stopped with HCl 1M (Merck) and optical density was measured at 450 nm using a SpectraMax microplate spectrophotometer . The IgG level in each sample was calculated interpolating the mean optical density value of the sample on a linear regression graphic of recombinant IgG (ref. 15260) standard curve dilution (ranging from 0.500 to 0.031 µg/mL) performed using the same conditions described above .Differences in percentages of B cells, B cell subsets, and ELISA were analyzed using the Kruskal-Wallis test. Graphs were produced by GraphPad Prism version 8.0 for Mac . The statistical significance level was set at p<0.05; p<0.005; p<0.0005.To study peripheral B cell subpopulations, initially, lumps and monocytes were excluded using the parameters of frontal dispersion measured by area , versus frontal dispersion measured by height . Next, the lymphocyte region was selected by FSC-A and lateral dispersion measured by SSC-A area (side scatter), and in this region B cells (CD3-CD19+) were detected. The B cell subpopulation gate strategies were applied to different leprosy manifestations and uninfected subjects. No difference between the different clinical forms and uninfected subjects was observed  effector cells . Our resUnswitched memory B cells (CD19+CD27+IgD+) are important in the first line of defense against infections because of the quick production of low-affinity IgM . Figure We did not observe differences in switched memory B cells between the different clinical profiles of the disease , 1539 in MB (range _ 3179), 319.7 in PB (IQ range _ 419.5), and 438.5 in ENL (range _ 589.4). Despite the relatively low number of analyzed donors, a significantly higher level of circulating IgG in patients with MB leprosy compared to PB (p=0.0004) and patients with ENL (p=0.0164) was observed  antibodies in patients with leprosy show a direct correlation with bacterial load suggesting that IgG1 B-cell responses may be surrogate markers of disease progression, although the role of B cells in the different clinical forms of the disease needs to be elucidated. There are still only few reports about the role of B cells in active leprosy lesions in different spectral forms of the disease, although there are several evidences of the involvement of B cells not only in the onset of reactional episodes but also in the course of non-reactional leprosy.Fabel and colleagues  have sugWe acknowledge that one limitation of the present study is the absence of functional analysis but a previous morphometric analysis of B cells and plasma cells in ENL demonstrated a positive correlation with CD3-positive pan T cells in the biopsy and a negative correlation with T regulatory – T cell ratio .Negera and colleagues reported that mature and memory B cells on patients with MB leprosy have no different frequencies from the ones in ENL . However2, leukotriene B4, lipoxin A4) when compared to PB, and these substances can inhibit B cell activation . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.ON and MG designed the analyses, collected, and analyzed data, and wrote the manuscript. VF and NC contributed with data collection. RR-d-S and JL contributed with analysis tools. RP, GP, and MP designed the analyses and supervised experimental work. CM supervised and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by The New York Community Trust/Heiser Program for Research on Leprosy and The National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIAID/NIH) under the award number RO1AI129835.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N = 433; Female: 58%; Mage = 23.51 [SD = 4.18]). The FCV-19S demonstrated a unidimensional factor structure and acceptable internal consistency (α = 0.87), Omega (ω = 0.88) and the greatest lower bound (GLB = 0.90) reliabilities. In addition, discriminant validity was demonstrated when FCV-19S items loaded separately from ordinary fear. The FCV-19S can be used as a measure of COVID-19-related fear amongst black South African university students.Coronavirus disease 2019 (COVID-19) has spread widely leading to a global public health crisis of a pandemic proportion. Whilst infection rates tend to fluctuate in South Africa, COVID-19 remains a life-threatening disease with the capacity to wreak fear and concern. The present study evaluated the psychometric qualities of the Fear of COVID-19 Scale (FCV-19S) amongst black South African university students ( Coronavirus disease 2019 (COVID-19) has spread widely leading to a global public health crisis. Aside from large-scale deaths, one of its consequences has been mental health problems  dimensionality, (2) discriminant validity and (3) reliability.age = 23.51, standard deviation [SD] = 4.18) was used for the study. The sample was predominantly constituted by undergraduate students (88%) across the faculty of Humanities, whilst 76% of the participants resided in a rural area  that were collected around the same time, although data set 1 does not have all the measures covered in data set 2. Data were collected by two different research groups using the same data collection media, during the same period and utilising the same student population.All participants consented to participation in the study before completing the questionnaire. Participants were recruited online using class registers. The registers were simply used to direct the communication to the potential respondents because they contained student-number formulated e-mail addresses (i.e. a university-generated e-mail address that only contains a student number and not the student’s name). They were directed to the website where the study questionnaire, designed using Google Forms, was posted. The survey was only in English and required between 20 and 30 min to complete. The total sample consists of two data sets  was used to examine the factor structure of the FCV-19S using confirmatory factor analysis (CFA). The CFA analyses (of the one-factor model consistently found in the literature) were conducted with the weighted least squares mean and variance adjusted (WLSMV) estimator for ordinal data in Mplus 7.4  and the physical response factor . The bi-factor model incorporated a general fear factor and two orthogonal factors .Two data sets were used to conduct the main analyses. A combined data set (n = 231) was used to evaluate the discriminant validity of the measure using exploratory factor analysis (EFA). The dimensionality of the FCV-19S and J-5FS items under EFA was estimated with maximum likelihood using principal axis factoring (PAF) with varimax rotation. Factor selection was performed using parallel analysis (PA).Following CFA, only data set 2  reliability estimates of the unidimensional scale of the FCV-19S were examined.Ethical compliance was approved by the Turfloop Research Ethics Committee of the University of Limpopo, reference number: TREC/375/2020: IR.Item mean, standard deviations and normality of the FCV-19S were examined. The univariate skewness and kurtosis for each of all the seven items of the scale are within the normal range of –1.5 to 1.5 see .X2 = 51.637, degrees of freedom [df] = 14, p < 0.001, TLI = 0.975, CFI = 0.983, SRMR = 0.070, RMSEA = 0.079, with a 90% CI [0.057–0.102]) and all parameters were viable. Two alternative models were also tested: two-factor model  and bi-factor model . Whilst the bi-factor model appears on the basis of fit indices to have a good fit, the model is however a poor model because of the fact that not all model parameters were viable (i.e. items 2 and 4 load poorly on the first factor). As such the overall results did not support the two alternative models.Results of CFA conducted on the FCV-19S revealed a well-fitting model see  to the dX2 = 1410.98, df = 21, p < 0.001) indicating that the data were suitable for EFA. Principal axis factoring produced a two-factor solution. The FCV-19S and the J-5FS were loaded and factor analysed together to establish discriminant validity. The Kaiser–Meyer–Olkin (KMO) measure was 0.86 showing that the sample size used in the study was adequate for EFA Field, , whilst The reliability estimates of the FCV-19S were acceptable .This study set out to evaluate the psychometric properties of the FCV-19S in the South African context, using a predominantly black African student population in the Limpopo province. The CFA results lend support to a unidimensional factor structure of the scale (Ahorsu et al., In spite of the FCV-19S being unidimensional, there is a pattern of response to the items where endorsements of items 3, 6 and 7 are comparatively low when contrasted with the scores of the remaining items. The pattern was observed in mean scores reported by studies such as Elemo et al. , GiordanThe reliability estimate of the FCV-19S is comparable with those obtained in many other studies from different geographic contexts (cf. Elemo et al., The limitation of the study is that the sample was not randomly drawn and therefore may not likely be completely representative of the targeted study population. There are more students domiciled in a rural area and they were over-represented in this sample. The study has to be replicated in a different research site and with non-student samples to confirm the results. Additional psychometric properties that could offer more validity evidence for the FCV-19S, such as predictive validity, convergent validity and measurement invariance, were not assessed.This study established that the FCV-19S can be used as a unidimensional measure of COVID-19-related fear amongst university students in South Africa. It is also reliable. The FCV-19S has also been shown to be a construct distinct from ordinary fear. Its scores will assist with messaging pertaining to COVID-19 prevention.


Colorectal cancer (CRC) begins when normal cells turn out of balance, and a tumor is formed in the lining of the colon or rectum. Cytotoxic T-lymphocyte protein 4 (CTLA-4) is a potent molecule that could inhibit T cell activation. Here, we analyzed this molecule in the tissue samples and cell lines of colorectal cancer to reveal the mechanism of this inhibitory molecule in CRC. Our result showed an increasing trend of CTLA-4 in tissues and cell lines. Finally, capecitabine as an approved drug in CRC could suppress this inhibitory molecule. It can be concluded that the inhibition of this inhibitory molecule can re-active the immune cells, especially T cells, in CRC patients, which boosts the immune cells against the tumor. Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory immune checkpoint that can be expressed in tumor-infiltrating lymphocytes and colorectal cancer (CRC) cells. This immune checkpoint can attenuate anti-tumoral immune responses and facilitate tumor growth and metastasis. Although capecitabine is an effective chemotherapeutic agent for treating CRC, its effect on the tumoral CTLA-4 expression remains unclear. In the current research, we applied the GSE110224 and GSE25070 datasets to characterize CTLA-4 expression in CRC patients. Then, we analyzed CTLA-4 expression in CRC samples, HT-29, HCT-166, and SW480 cell lines using real-time PCR. Our bioinformatic results have highlighted the overexpression of CTLA-4 in the CRC tissues compared to the adjacent non-tumoral tissues. Our in vitro studies have indicated that SW480 cells can substantially overexpress CTLA-4 compared to HT-29 and HCT 116 cells. In addition, capecitabine can remarkably downregulate the expression of CTLA-4 in SW480 cells. Collectively, capecitabine can inhibit the expression of CTLA-4 in CRC cells and might bridge the immunotherapy approaches with chemotherapy. CRC is responsible for the third cause of cancer-related death among men and the second cause of cancer-related death among women . AdditioInhibitory immune checkpoints can inhibit the development of anti-tumoral immune responses and pave the way for tumor proliferation and metastasis. CTLA-4/CD80, CD86 axis, which can be established between immune cells and tumoral cells, has been implicated in attenuating the development of anti-tumoral immune responses in the tumor microenvironment. Indeed, the satisfactory results of CTLA-4 inhibitors in rejecting various tumors have translated into the FDA’s approval to treat patients with cancers . ConsistChemotherapy has been one of the mainstream approaches for treating patients with cancers. As one of the effective chemotherapeutic agents for treating CRC, capecitabine is designed to inhibit the fast-paced tumor proliferation via interrupting the cell cycle and inducing apoptosis ,6. In adThis study aimed to use clinical specimens and bioinformatic tools to assess the CTLA-4 expression in cancer and adjacent samples. Moreover, we intended to investigate the effect of capecitabine on CTLA-4 expression in the SW480 cells. The results of this study might further our knowledge of capecitabine on the tumoral CTLA-4 expression and might bridge the immunotherapy approaches with chemotherapy.https://www.ncbi.nlm.nih.gov/geo/ (accessed on 1 May 2021)). The GSE110224 dataset was based on GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array with 34 samples (17 patients with CRC and 17 adjacent samples). The GSE25070 dataset, based on the Illumina HumanRef-8 v3.0 expression bead chip, included 26 CRC samples and 26 adjacent non-tumor colorectal tissue samples. The raw data were corrected and quantile-normalized with the affy package of R 3.4.1 in Bioconductor [Data preprocessing is a data mining technique that involves transforming raw data into an understandable format. The GSE110224 and GSE25070 microarray datasets were downloaded from the Gene Expression Omnibus (GEO) database (onductor . The annhttps://xenabrowser.net/ (accessed on 1 May 2021)) Then, CTLA-4 expression in HT-29, HCT 116, and SW480 cells was obtained from the Cancer Cell Line Encyclopedia (CCLE) datasets using the Xena browser. Finally, survival analysis of the CTLA-4 gene was demonstrated in Kaplan–Meier survival curves.For initial validation of CTLA-4 expression in primary tumors, normal solid tissue, recurrent tumor, and metastatic tumors, data were extracted from the TCGA-COAD database using the UCSC Cancer Browser . All participants signed written informed consent before the surgery. Eighteen samples of colorectal and adjacent non-tumoral samples were included in the study. Inclusion criteria for this study were individuals with age ≥ 30 years, without a history of inflammatory diseases, without a known hereditary predisposition to CRC, or other overwhelming disorders. All included CRC patients were histopathologically confirmed by experienced pathologists. The fresh samples were collected and transported in a nitrogen tank from the operation room to the laboratory. Diethyl pyruvate carbonate (DEPC) was used to remove RNase.2 at 37 °C.Three CRC cell lines, i.e., HT-29, HCT 116, and SW480, were purchased from the Pasture Institute of Iran. These cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin  in an incubator with 5% CO4) were seeded in six-well plates and incubated at 37 °C in 5% CO2. After 24 h, the cells were harvested using trypsin/EDTA, centrifuged at 1500 g for 15 min, and prepared for RNA extraction, cDNA synthesis, and real-time PCR.The HT-29, HCT 116, and SW480 cells -2,5-diphenyl-2H-tetrazolium bromide (MTT) was added to the wells for 4 h (5 mg/mL). After removing the medium, 150 μL dimethyl sulfoxide (DMSO) was added to each well, and the plate was shaken for 10 min. A microplate reader  at a test wavelength of 570 nm and a reference wavelength of 630 nm was used to obtain the information.The 15 × 10According to the manufacturer’s protocol, the whole RNA from colorectal tissue and marginal samples was extracted using Trizol reagent (RiboEx). Before RNA extraction, whole tissues were placed in a tissue homogenizer on Trizol and squashed using a tissue homogenizer with Serrated-Pestle (Grinded Vessel) on ice several times. The cDNA synthesis kit  was used to synthesize cDNA.A 2X Master Mix with high ROX (Biofact) was used for real-time PCR analysis. Three runs were conducted for each experiment. The PCR condition was as follows: initial denaturation for 13 min at 95 °C; 45 cycles of denaturation for 13 s, 95 °C; annealing, 30 s, 60 °C; elongation, 20 s, 72 °C. The specific primer sequences were used as follows: CTLA-4  and GAPDH . In the current research, GAPDH was used as an internal control.After real-time PCR analysis, electrophoresis was carried out to detect the CTLA-4 and GAPDH gene expressions in the SW480 cells before and after treatment with capecitabine; 3% agarose gel was used for this purpose.p < 0.05 was considered a criterion for statistical significance between groups.GraphPad Prism 6.0  was used to analyze the data. The paired t-test was used to compare the data with the controls, and one-way ANOVA was used to determine if there were statistically significant differences in the comparison of multiple groups. R version 3.4.1 was used for the in-silico analysis. We performed quantile normalization to reduce the effects of technical noises. Probe ID conversion and averaging probes were conducted as mentioned in the previous study . Figure p-value = 1.91 × 10−6) for GSE25070 and 1.35  for GSE110224 dataset.CTLA-4 expression was evaluated in two datasets, i.e., GSE110224 and GSE25070. We used heatmap analysis to describe the values of CTLA-4 in the samples of these two datasets. The results demonstrated significant expression patterns in the samples C. As shohttps://xenabrowser.net/ (accessed on 1 May 2021)). As shown in p = 2.1 × 10−7). Our results showed that CTLA-4 gene expression was upregulated in the primary CRC compared to non-tumoral samples.We used RNA-sequencing data for the initial analysis of the CTLA-4 gene expression in CRC and non-tumoral samples. The clinicopathological and survival data of patients with primary CRC were obtained from the UCSC Xena browser (p = 0.059)  C.Eighteen patients with CRC were enrolled in the current study. (−ΔΔ CT) method. Although our results showed a trend in the increased expression of CTLA-4 in the CRC tissues, this change was not statistically significant (p = 0.202)  .p < 0.0001 and p < 0.001, respectively)  .Our results showed that capecitabine could decrease the viability of SW480 cells in a dose-dependent manner. The MTT results indicated that 289.7 ng/mL was the half-maximal inhibitory concentration (IC50) of capecitabine for SW480 cells. This value was used for conducting experiments to determine the effect of capecitabine on the CTLA-4 gene expression in the SW480 cells .p < 0.0001)  A. The pr 0.0001) B.Despite remarkable advances in understating CRC biology and their interaction with the tumor microenvironment, the current approaches have not brought a treatment with the highest response rate and the lowest side effects for affected patients. A better understanding of the interaction between tumor microenvironment with CRC cells might pave the way for developing a more efficient therapy for CRC patients . Our biop-value = 0.001, and HR = 2.53, 95%, CI: 1.23–4.95, p-value = 0.01, respectively) [p-value > 0.05). Our bioinformatic results have failed to depict a statistically significant figure for the prognostic value of CTLA-4 for determining the overall survival of CRC patients .Initial studies have shown that CTLA-4 can be expressed in tumor-infiltrating lymphocytes, leading to the attenuation of anti-tumoral immune responses. However, recent findings have demonstrated that CTLA-4 can also be expressed on tumoral cells and shield tumor cells from immune responses ,13. Liu ctively) . AlthougAlthough identifying immune checkpoint axes have opened up a new era for cancer therapy, monotherapy with immune checkpoint inhibitors has not shown the efficacy as would be expected. The disparity between response rates of immunotherapy approaches between patients has not been ignorable. Although the advances in the single-cell sequencing techniques might bring novel solutions for improving the response rates of immunotherapy approaches, one of the currently accepted approaches is the combination of chemotherapy approaches with immune checkpoint inhibitors ,17. TherOur study has several strengths. First, our study has used bioinformatics, clinical samples, and cellular studies to address the research question. Second, the current study aimed to provide a blueprint for further investigations regarding the combination value of immune checkpoint inhibitors and capecitabine. However, the present study has several limitations as well. First, our small sample size and lack of an organized registration system did not allow us to include more CRC patients and provide survival analysis. Second, we could not measure the protein expression of CTLA-4 following treating the SW480 cells with capecitabine.Based on GSE110224 and GSE25070 datasets, we have found that CTLA-4 expression is substantially upregulated in CRC tissues compared to adjacent non-tumor samples. Although the expression of CTLA-4 has been remarkably different among primary CRC, non-tumoral samples, recurrent tumors, and metastatic tumors. Our results have failed to demonstrate a significant prognostic value for CTLA-4 expression for determining the overall survival of CRC patients. Based on our eighteen CRC samples, we have found a trend in the increased expression of CTLA-4 in CRC tissues compared to the adjacent non-tumoral tissues; however, this change was not statistically significant. We have found that SW480 cells overexpress CTLA-4 compared to HT-29 and HCT 116 cells, and treating SW480 cells with the IC50 of capecitabine, 289.7 ng/mL, can considerably decrease CTLA-4 expression in SW480 cells. Further in vivo analysis and animal model studies are needed to reveal the exact effect of capecitabine on the CTLA-4 expression in CRC patients.